Anti-apoptotic BCL-2 family members regulate hematopoiesis and when dysregulated contribute to cancer, immunodeficiency, and autoimmunity. MCL-1 is absolutely required for cell survival at multiple stages of hematopoietic development. In contrast, genetic ablation of Bcl-2 or Bcl-X has revealed specific roles in promoting hematopoietic survival. Why MCL-1 plays such a critical role in maintaining survival and why the concomitant endogenous expression of other anti-apoptotic regulators cannot compensate for MCL-1 loss has remained unresolved. Therefore, the long term goal of my laboratory is to understand how MCL-1 functions and is regulated during hematopoietic development and survival. To understand the requirement for MCL-1 in development, we have recently identified that it localizes to both the outer mitochondrial membrane, where it MCL-1 binds and sequesters pro-apoptotic molecules, and to the mitochondrial inner membrane as an N- terminally truncated form. The contribution of both of these localizations to MCL-1's function is unclear. The objective of this proposal is to dissect the functional roles of the two mitochondrial forms of MCL-1 and to assess the individual roles of these localizations in promoting cellular survival. Our central hypothesis is that the outer membrane form of MCL-1 acts like classical BCL-2 family members to antagonize pro-apoptotics, while the inner membrane form of MCL-1 regulates cell survival by modulating mitochondrial function. The studies we propose will reveal a previously unrecognized role for MCL-1 and shed light onto a new mechanism by which an anti-apoptotic BCL-2 family member promotes mitochondrial function. Aim 1: Define the role(s) for the different mitochondrial forms of MCL-1 in regulating hematopoiesis. MCL-1 exhibits a profound requirement during many stages of hematopoiesis, but it is unclear why. Aim 2: Define the role(s) of inner mitochondrial MCL-1 in regulating apoptosis. Our preliminary studies indicate that only the outer mitochondrial form of MCL-1 is capable of binding and sequestering pro-apoptotic molecules. However, it is unclear whether the inner mitochondrial form of MCL-1 may alter response to death stimuli. Therefore, we will investigate how the different forms of MCL-1 contribute to its classical anti-apoptotic nature. Aim 3: Identify how MCL-1 regulates mitochondrial physiology. Our preliminary studies indicate that Mcl-1-deficiency results in abnormalities in mitochondrial physiology even when cells are not undergoing apoptosis. My laboratory has made many of the seminal findings defining the role of MCL-1 in promoting survival during hematopoiesis. Furthermore, we are uniquely positioned to successfully perform these studies as we have identified the novel localization, generated MCL-1 mutants that can dissect the different forms of MCL-1, and have extensive experience in MCL-1 biology. At the end of this study, we will have illuminated a previously unrecognized role for MCL-1 in promoting mitochondrial function.